Nucleotide sequences which code for the cma gene

ABSTRACT

The invention relates to a genetically modified coryneform bacterium, the cma gene of which is amplified, and an isolated polynucleotide which codes for cyclopropane-mycolic acid synthase from coryneform bacteria, and also a method for the fermentative preparation of L-amino acids with amplification of the cma gene in the bacteria and the use of the polynucleotide as a primer or hydridization probe.

RELATED APPLICATION DATA

[0001] This application is a continuatin-in-Part of co-pending U.S.patent application Ser. No. 09/577,857 filed May 25, 2000, whichapplication claims priority under 35 U.S.C. §119 from German PatentAppln. No. 10021832.6, filed in Germany on May 4, 2000. Theabove-identified U.S. patent application and German patent applicationare entirely incorporated herein by reference.

[0002] The invention provides genetically modified coryneform bacteria,nucleotide sequences which code for cyclopropane-mycolic acid synthaseand a method for the fermentative preparation of amino acids, inparticular L-lysine and L-glutamate, using coryneform bacteria in whichthe cma gene, which codes for cyclopropane-mycolic acid synthase, isamplified. All references cited herein are expressly incorporated byreference. Incorporation by reference is also designated by the term“I.B.R.” following any citation.

PRIOR ART

[0003] Amino acids, in particular L-lysine and L-glutamate, are used inhuman medicine, in the pharmaceuticals industry, in the foodstuffsindustry, but in particular in animal nutrition.

[0004] It is known that amino acids are produced by fermentation fromstrains of coryneform bacteria, in particular Corynebacteriumglutamicum. Because of their great importance, work is constantly beingundertaken to improve the preparation methods. Improvements to themethods can relate to fermentation measures, such as e. g. stirring andsupply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up tothe product form by e. g. ion exchange chromatography, or the intrinsicoutput properties of the microorganism itself.

[0005] Methods of mutagenesis, selection and mutant selection are usedto improve the output properties of these microorganisms. Strains whichare resistant to antimetabolites, such as e. g. the lysine analogueS-(2-aminoethyl)-cysteine, or are auxotrophic for metabolites ofregulatory importance and produce L-amino acids, such as e. g. L-lysineor L-glutamate, are obtained in this manner.

[0006] Methods of the recombinant DNA technique have also been employedfor some years for improving the strain of Corynebacterium strains whichproduce amino acids, by amplifying individual amino acid biosynthesisgenes and investigating the effect on the amino acid production. Reviewarticles in this context are to be found, inter alia, in Kinoshita(“Glutamic Acid Bacteria”, in: Biology of Industrial Microorganisms,Demain and Solomon (Eds.) I.B.R., Benjamin Cummings, London, UK, 1985,115-142), Hilliger (BioTec 2, 40-44 (1991)) I.B.R., Eggeling (AminoAcids 6:261-272 (1994)) I.B.R., Jetten and Sinskey (Critical Reviews inBiotechnology 15, 73-103 (1995)) I.B.R. and Sahm et al. (Annuals of theNew York Academy of Science 782, 25-39 (1996)) I.B.R.

OBJECT OF THE INVENTION

[0007] The object of the present invention was to provide new aids forimproved fermentative preparation of amino acids, in particular L-lysineand L-glutamate.

[0008] This object is achieved by a genetically modified coryneformbacterium, the cma gene of which, which codes for cyclopropane-mycolicacid synthase, is amplified.

[0009] Amino acids, in particular L-lysine and L-glutamate, are used inhuman medicine, in the pharmaceuticals industry, in the foodstuffsindustry, and in particular in animal nutrition. There is therefore ageneral interest in providing new improved methods for the preparationof amino acids, in particular L-lysine and L-glutamate.

[0010] When L-lysine or lysine and L-glutamate or glutamate arementioned in the following, not only the bases but also the salts, suchas e. g. lysine monohydrochloride or lysine sulfate, are also meant bythis.

SUMMARY OF THE INVENTION

[0011] The new DNA sequence of C. glutamicum which codes for the cmagene and which as a constituent of the present invention is SEQ ID NO 1and related sequences. The amino acid sequence of the corresponding geneproduct of the cma gene has furthermore been derived from the presentDNA sequence. The resulting amino acid sequence of the cma gene productis SEQ ID NO 2 and related sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention will be further understood with referenceto the drawing offered here for illustration only and not in limitationof this invention.

[0013]FIG. 1: Map of the plasmid pJC1cma

DETAILED DESCRIPTION OF THE INVENTION

[0014] The invention provides a genetically modified coryneformbacterium, in which its cma gene, which codes for cyclopropane-mycolicacid synthase, is amplified.

[0015] The term “amplification” in this connection describes theincrease in intracellular activity of one or more enzymes in amicroorganism which are coded by the corresponding DNA.

[0016] Amplification can be achieved with the aid of variousmanipulations of the bacterial cell.

[0017] To achieve an amplification, in particular an over-expression,the number of copies of the corresponding genes can be increased, apotent promoter can be used, or the promoter and regulation region orthe ribosome binding site upstream of the structural gene can bemutated. Expression cassettes which are incorporated upstream of thestructural gene act in the same way. By inducible promoters, it isadditionally possible to increase the expression in the course offermentative L-lysine or L-glutamate production. It is also possible touse a gene which codes for a corresponding enzyme with a high activity.The expression is likewise improved by measures to prolong the life ofthe m-RNA. Furthermore, the enzyme activity is also increased overall bypreventing the degradation of the enzyme. These measures can optionallyalso be combined as desired.

[0018] The microorganisms which the present invention provides canprepare L-amino acids, in particular L-lysine and L-glutamate, fromglucose, sucrose, lactose, fructose, maltose, molasses, starch,cellulose or from glycerol and ethanol. They can be representatives ofcoryneform bacteria, in particular of the genus Corynebacterium. Of thegenus Corynebacterium, there may be mentioned in particular the speciesCorynebacterium glutamicum, which is known among experts for its abilityto produce L-amino acids.

[0019] Suitable strains of the genus Corynebacterium, in particular ofthe species Corynebacterium glutamicum, are, for example, the knownwild-type strains

[0020]Corynebacterium glutamicum ATCC13032

[0021]Corynebacterium acetoglutamicum ATCC15806

[0022]Corynebacterium acetoacidophilum ATCC13870

[0023]Corynebacterium thermoaminogenes FERM BP-1539

[0024]Corynebacterium melassecola ATCC17965

[0025]Brevibacterium flavum ATCC14067

[0026]Brevibacterium lactofermentum ATCC13869 and

[0027]Brevibacterium divaricatum ATCC14020

[0028] and L-lysine-producing mutants or strains prepared therefrom,such as, for example

[0029]Corynebacterium glutamicum FERM-P 1709

[0030]Brevibacterium flavum FERM-P 1708

[0031]Brevibacterium lactofermentum FERM-P 1712

[0032]Corynebacterium glutamicum FERM-P 6463

[0033]Corynebacterium glutamicum FERM-P 6464 and

[0034]Corynebacterium glutamicum DSM5715.

[0035] The present invention also provides an isolated polynucleotidefrom coryneform bacteria, comprising a polynucleotide sequence chosenfrom the group consisting of

[0036] a) polynucleotide which is homologous to the extent of at least70% with a polynucleotide which codes for a polypeptide which comprisesthe amino acid sequence of SEQ ID No. 2,

[0037] b) polynucleotide which codes for a polypeptide which comprisesan amino acid sequence which is homologous to the extent of at least 70%with the amino acid sequence of SEQ ID No. 2,

[0038] c) polynucleotide which is complementary to the polynucleotidesof a) or b), and

[0039] d) polynucleotide comprising at least 15 successive nucleotidesof the polynucleotide sequence of a), b) or c).

[0040] In the context of the present Application, a polynucleotidesequence is “homologous” to the sequence according to the invention ifit coincides in its base composition and sequence with the sequenceaccording to the invention to the extent of at least 70%, preferably atleast 80%, particularly preferably at least 90%. According to thepresent invention, a “homologous protein” is to be understood asproteins which have an amino acid sequence which coincide with the aminoacid sequence coded by the cma gene (SEQ ID No. 1) to the extent of atleast 70%, preferably at least 80%, particularly preferably at least90%, “coincide” being understood as meaning that either thecorresponding amino acids are identical or they are amino acids whichare homologous to one another. Those amino acids which correspond intheir properties, in particular in respect of charge, hydrophobicity,steric properties etc., are called “homologous amino acids”.

[0041] The invention also provides a polynucleotide as described above,this preferably being a DNA which is capable of replication, comprising:

[0042] (i) the nucleotide sequence shown in SEQ ID no. 1, or

[0043] (ii) at least one sequence which corresponds to sequence (i) inthe context of the degeneration of the genetic code, or

[0044] (iii) at least one sequence which hybridizes with the sequencecomplementary to sequence (i) or (ii), and optionally

[0045] (iv) mutations of neutral function in (i) which lead to the sameor a homologous amino acid.

[0046] The relative degree of substitution or mutation in thepolynucleotide or amino acid sequence to produce a desired percentage ofsequence identity can be established or determined by well-known methodsof sequence analysis. These methods are disclosed and demonstrated inBishop, et al. “DNA & Protein Sequence Analysis (A Practical Approach”),Oxford Univ. Press, Inc. (1997) I.B.R. and by Steinberg, Michael“Protein Structure Prediction” (A Practical Approach), Oxford Univ.Press, Inc. (1997) I.B.R. Hybridization of complementary sequences canoccur at varying degrees of stringency. Sambrook et al.: MolecularCloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989)I.B.R.

[0047] Hybridization of complementary sequences can occur at varyingdegrees of stringency. Sambrook et al.: Molecular Cloning, A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1989) I.B.R. Instructionsfor identifying DNA sequences by means of hybridization can be found bythe expert, inter alia, in the handbook “The DIG System Users Guide forFilter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany,1993) I.B.R. and in Liebl et al. (International Journal of SystematicBacteriology (1991) 41: 255-260) I.B.R.

[0048] Comprehensive descriptions can be found in known textbooks ofgenetics and molecular biology, such as e. g. that by Hagemann(“Allgemeine Genetik” [General Genetics], Gustav Fischer Verlag,Stuttgart, 1986) I.B.R.

[0049] Possible mutations are transitions, transversions, insertions anddeletions. Depending on the effect of the amino acid exchange on theenzyme activity, missense mutations or nonsense mutations are referredto. Insertions or deletions of at least one base pair in a gene lead toframe shift mutations, as a consequence of which incorrect amino acidsare incorporated or translation is interrupted prematurely. Deletions ofseveral codons typically lead to a complete loss of the enzyme activity.

[0050] Instructions on generation of such mutations are prior art andcan be found in known textbooks of genetics and molecular biology, suchas e. g. the textbook by Knippers (“Molekulare Genetik” [MolecularGenetics], 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995)I.B.R., that by Winnacker (“Gene und Klone” [Genes and Clones], VCHVerlagsgesellschaft, Weinheim, Germany, 1990) I.B.R. or that by Hagemann(“Allgemeine Genetik” [General Genetics], Gustav Fischer Verlag,Stuttgart, 1986) I.B.R.

[0051] The invention also provides a vector containing one of the statedpolynucleotides and coryneform bacteria acting as host cell whichcontain the vector or in which the cma gene is amplified.

[0052] The invention also provides

[0053] a polynucleotide which is capable of replication and comprisesthe nucleotide sequence SEQ ID No. 1, or consists of it,

[0054] a polynucleotide sequence which codes for a polypeptide whichcomprises the amino acid sequence SEQ ID No. 2, or consists of it,

[0055] a vector containing the DNA sequence of C. glutamicum which codesfor the cma gene, contained in the vector (plasmid) pJC1cma, depositedin Corynebacterium glutamicum under number DSM 13248,

[0056] and coryneform bacteria serving as the host cell, which containthe vector or in which the cma gene is amplified.

[0057] The invention also provides polynucleotides which comprise thecomplete gene with the polynucleotide sequence corresponding to SEQ IDNo. 1 or fragments thereof, and which are obtainable by screening bymeans of hybridization of a corresponding gene library with a probewhich comprises the sequence of the polynucleotide mentioned, accordingto SEQ ID No. 1, or a fragment thereof, and isolation of the DNAsequence mentioned.

[0058] Polynucleotide sequences according to the invention are alsosuitable as hybridization probes for RNA, cDNA and DNA, in order toisolate, in the full length, cDNA which codes for cyclopropane-mycolicacid synthase and to isolate those cDNA or genes which have a highsimilarity of with the sequence of the cyclopropane-mycolic acidsynthase gene.

[0059] Polynucleotide sequences according to the invention arefurthermore suitable as primers for the polymerase chain reaction (PCR),for the preparation of DNA which codes for cyclopropane-mycolic acidsynthases.

[0060] Such oligonucleotides which serve as probes or primers cancomprise more than 30, preferably up to 30, particularly preferably upto 20, especially preferably at least 15 successive nucleotides.Oligonucleotides which have a length of at least 40 or 50 nucleotidesare also suitable.

[0061] “Isolated” means separated out of its natural environment.

[0062] “Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

[0063] “Polypeptides” is understood as meaning peptides or proteinswhich comprise two or more amino acids bonded via peptide bonds.

[0064] The polypeptides according to the invention include a polypeptideaccording to SEQ ID No. 2, in particular those with the biologicalactivity of cyclopropane-mycolic acid synthase and also those which arehomologous to the extent of at least 70% with the polypeptide accordingto SEQ ID No. 2, preferably to the extent of at least 80%, andparticularly preferably those which are homologous to the extent of atleast 90 to 95% with the polypeptide according to SEQ ID no. 2, and havethe activity mentioned.

[0065] The invention moreover provides a method for the fermentativepreparation of amino acids, in particular L-lysine and L-glutamate,using coryneform bacteria which in particular already produce an aminoacid, and in which the nucleotide sequences which code for the cma geneare amplified, in particular over-expressed.

[0066] The cma gene of C. glutamicum which codes forcyclopropane-mycolic acid synthase is described for the first time inthe present invention.

[0067] To isolate the cma gene or also other genes of C. glutamicum, agene library of this microorganism was first set up in E. coli. Thesetting up of gene libraries is described in generally known textbooksand handbooks. The textbook by Winnacker: Gene und Klone, EineEinfüthrung in die Gentechnologie [Genes and Clones, An Introduction toGenetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990) I.B.R. orthe handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual(Cold Spring Harbor Laboratory Press, 1989) I.B.R. may be mentioned asan example. A well-known gene library is that of the E. coli K-12 strainW3110 set up in λ vectors by Kohara et al. (Cell 50, 495-508 (1987))I.B.R. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996)I.B.R. describe a gene library of C. glutamicum ATCC13032, which was setup with the aid of the cosmid vector SuperCos I (Wahl et al., 1987,Proceedings of the National Academy of Sciences USA, 84:2160-2164)I.B.R. in the E. coli K-12 strain NM554 (Raleigh et al., 1988, NucleicAcids Research 16:1563-1575) I.B.R. Börmann et al. (MolecularMicrobiology 6(3), 317-326)) (1992)) I.B.R. in turn describe a genelibrary of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn andCollins, Gene 11, 291-298 (1980)) I.B.R. To prepare a gene library of C.glutamicum in E. coli it is also possible to use plasmids such as pBR322(Bolivar, Life Sciences, 25, 807-818 (1979)) I.B.R. or pUC9 (Vieira etal., 1982, Gene, 19:259-268) I.B.R. Suitable hosts are, in particular,those E. coli strains which are restriction- andrecombination-defective. An example of these is the strain DH5αcmcr,which has been described by Grant et al. (Proceedings of the NationalAcademy of Sciences USA, 87 (1990) 4645-4649) I.B.R. The long DNAfragments cloned with the aid of cosmids can then in turn be subclonedand subsequently sequenced in the usual vectors which are suitable forsequencing, such as is described e. g. by Sanger et al. (Proceedings ofthe National Academy of Sciences of the United States of America,74:5463-5467, 1977) I.B.R.

[0068] The new DNA sequence of C. glutamicum which codes for the cmagene and which, as SEQ ID No. 1, is a constituent of the presentinvention, was obtained in this manner. The amino acid sequence of thecorresponding protein has moreover been derived from the present DNAsequence by the methods described above. The resulting amino acidsequence of the cma gene product is shown in SEQ ID No. 2.

[0069] Coding DNA sequences which result from SEQ ID No. 1 by thedegeneracy of the genetic code are also a constituent of the invention.In the same way, DNA sequences which hybridize with SEQ ID No. 1 orparts of SEQ ID No. 1 are a constituent of the invention. Conservativeamino acid exchanges, such as e. g. exchange of glycine for alanine orof aspartic acid for glutamic acid in proteins, are moreover known amongexperts as “sense mutations” which do not lead to a fundamental changein the activity of the protein, i.e. are of neutral function. It ismoreover known that changes on the N and/or C terminus of a proteincannot substantially impair the function thereof or can even stabilizethis. Information in this context can be found by the expert, interalia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987))I.B.R., in O'Regan et al. (Gene 77:237-251 (1989)) I.B.R., in Sahin-Tothet al. (Protein Sciences 3:240-247 (1994)) I.B.R., in Hochuli et al.(Bio/Technology 6:1321-1325 (1988)) I.B.R. and in known textbooks ofgenetics and molecular biology. Amino acid sequences which result in acorresponding manner from SEQ ID No. 2 are also a constituent of theinvention.

[0070] In the same way, DNA sequences which hybridize with SEQ ID No. 1or parts of SEQ ID No. 1 are a constituent of the invention. Finally,DNA sequences which are prepared by the polymerase chain reaction (PCR)using olognucleotide primers which result from SEQ ID NO. 1 are aconstituent of the invention. Such oligonucleotides typically have alength of at least 15 nucleotides.

[0071] Instructions for identifying DNA sequences by means ofhybridization can be found by the expert, inter alia, in the handbook“The DIG System Users Guide for Filter Hybridization” I.B.R. fromBoehringer Mannheim GmbH (Mannheim, Germany, 1993) I.B.R. and in Lieblet al. (International Journal of Systematic Bacteriology (1991) 41:255-260) I.B.R. Instructions for amplification of DNA sequences with theaid of the polymerase chain reaction (PCR) can be found by the expert,inter alia, in the handbook by Gait: Oligonukleotide synthesis: apractical approach (IRL Press, Oxford, UK, 1984) I.B.R. and in Newtonand Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany,1994) I.B.R.

[0072] In the work on the present invention, it has been found thatcoryneform bacteria produce amino acids, in particular L-lysine andL-glutamate, in an improved manner after amplification of the cma gene.

[0073] The genes or gene constructs under consideration can either bepresent in plasmids with a varying number of copies, or can beintegrated and amplified in the chromosome. Alternatively, anover-expression of the genes in question can moreover be achieved bychanging the composition of the media and the culture procedure.

[0074] Instructions in this context can be found by the expert, interalia, in Martin et al. (Bio/Technology 5, 137-146 (1987)) I.B.R., inGuerrero et al. (Gene 138, 35-41 (1994)) I.B.R., Tsuchiya and Morinaga(Bio/Technology 6, 428-430 (1988)) I.B.R., in Eikmanns et al. (Gene 102,93-98 (1991)) I.B.R., in European Patent Specification EPS 0 472 869I.B.R., in U.S. Pat. No. 4,601,893 I.B.R., in Schwarzer and Pühler(Bio/Technology 9, 84-87 (1991) I.B.R., in Reinscheid et al. (Appliedand Environmental Microbiology 60, 126-132 (1994)) I.B.R., in LaBarre etal. (Journal of Bacteriology 175, 1001-1007 (1993)) I.B.R., in PatentApplication WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993))I.B.R., in Japanese Laid-Open Specification JP-A-10-229891, in Jensenand Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) I.B.R.,in Makrides (Microbiological Reviews 60:512-538 (1996)) I.B.R. and inknown textbooks of genetics and molecular biology.

[0075] By way of example, the cma gene according to the invention wasover-expressed with the aid of plasmids.

[0076] Suitable plasmids are those which are replicated and expressed incoryneform bacteria. Numerous known plasmid vectors, such as e. g. pZ1(Menkel et al., Applied and Environmental Microbiology (1989) 64:549-554) I.B.R., pEKE×1 (Eikmanns et al., Gene 102:93-98 (1991)) orpHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) I.B.R. are based on thecryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such ase. g. those based on pCG4 (U.S. Pat. No. 4,489,160) I.B.R., or pNG2(Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990))I.B.R., or pAG1 (U.S. Pat. No. 5,158,891) I.B.R., can be used in thesame manner.

[0077] An example of a plasmid, with the aid of which the cma gene canbe over-expressed is pJC1cma (FIG. 1), which is based on the E. coli-C.glutamicum shuttle vector pJC1 (Cremer et al., 1990, Molecular andGeneral Genetics 220: 478- 480) I.B.R. and contains the DNA sequence ofCorynebacterium glutamicum which codes for the cma gene. It is containedin the strains ATCC 13032/pJC1cma and DSM5715/pJC1cma.

[0078] Plasmid vectors which are moreover suitable are those with theaid of which the method of gene amplification by integration into thechromosome can be used, as has been described, for example, byReinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)) I.B.R. for duplication or amplification of the hom-thrB operon.In this method, the complete gene is cloned in a plasmid vector whichcan replicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)) I.B.R., pK18mob or pK19mob (Schäfer et al., Gene 145,69-73 (1994)) I.B.R., pGEM-T (Promega corporation, Madison, Wis., USA),pCR2.1-TOPO (Shuman (1994) I.B.R. Journal of Biological Chemistry269:32678-84; U.S. Pat. No. 5,487,993) I.B.R., pCR®Blunt (Invitrogen,Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)) I.B.R. or pEM1 (Schrumpf et al, 1991, Journal ofBacteriology 173:4510-4516) I.B.R. The plasmid vector which contains thegene to be amplified is then transferred into the desired strain of C.glutamicum by conjugation or transformation. The method of conjugationis described, for example, by Schäfer et al. (Applied and EnvironmentalMicrobiology 60, 756-759 (1994)) I.B.R. Methods for transformation aredescribed, for example, by Thierbach et al. (Applied Microbiology andBiotechnology 29, 356-362 (1988)) I.B.R., Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989)) I.B.R. and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)) I.B.R. After homologousrecombination by means of a “cross over” event, the resulting straincontains at least two copies of the gene in question.

[0079] In addition, it may be advantageous for the production of aminoacids, in particular L-lysine and L-glutamate, to amplify orover-express one or more enzymes of the particular biosynthesis pathway,of glycolysis, of anaplerosis, of the citric acid cycle or of amino acidexport, in addition to the cma gene.

[0080] Thus, for example, for the preparation of L-lysine, one or moregenes chosen from the group consisting of

[0081] the dapA gene which codes for dihydrodipicolinate synthase (EP-B0 197 335 I.B.R.), or

[0082] the dapE gene which codes for succinyl diaminopimelatedesuccinylase, or

[0083] the lysC gene which codes for a feed-back resistant aspartatekinase (Kalinowski et al. (1990), Molecular and General Genetics 224,317-324 I.B.R.), or

[0084] the gap gene which codes for glyceraldehyde 3-phosphatedehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086I.B.R.), or

[0085] the tpi gene which codes for triose phosphate isomerase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086 I.B.R.), or

[0086] the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086 I.B.R.), or

[0087] the pyc gene which codes for pyruvate carboxylase (DE-A-19831609I.B.R.), or

[0088] the mqo gene which codes for malate-quinone oxidoreductase(Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)I.B.R.), or

[0089] the lysE gene which codes for lysine export (DE-A-195 48 222I.B.R.)

[0090] can be amplified, in particular over-expressed or amplified, atthe same time.

[0091] Furthermore, for example, for the preparation of L-glutamate, oneor more genes chosen from the group consisting of

[0092] the gdh gene which codes for glutamate dehydrogenase (DE:19907347.3 I.B.R.) and/or

[0093] the pyc gene which codes for pyruvate carboxylase(Peters-Wendisch et al. (1998), Microbiology 144: 915-927 I.B.R.)

[0094] can be amplified, in particular over-expressed or amplified, atthe same time.

[0095] In addition to amplification of the cma gene it may moreover beadvantageous for the production of L-lysine to attenuate

[0096] the pck gene which codes for phosphoenol pyruvate carboxykinase(DE 199 50 409.1, DSM 13047 I.B.R.) and/or

[0097] the pgi gene which codes for glucose 6-phosphate isomerase (U.S.Ser. No. 09/396,478, DSM 12969 I.B.R.)

[0098] at the same time.

[0099] In addition to amplification of the cma gene it may moreover beadvantageous for the production of L-glutamate to attenuate

[0100] the odhA gene which codes for α-ketoglutarate dehydrogenase (WO9534672 A1 951221* I.B.R.), or

[0101] the dtsR1 gene which codes for the DtsR1 protein (WO 952324 A1950831* I.B.R.), or

[0102] the dtsR2 gene which codes for the DtsR2 protein (WO 9902692A A1990121* I.B.R.)

[0103] at the same time.

[0104] In addition to over-expression of the cma gene it may moreover beadvantageous for the production of amino acids, in particular L-lysineand L-glutamate, to eliminate undesirable side reactions (Nakayama:“Breeding of Amino Acid Producing Micro-organisms”, in: Overproductionof Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press,London, UK, 1982 I.B.R.).

[0105] The microorganisms prepared according to the invention can becultured continuously or discontinuously in the batch method (batchculture) or in the fed batch (feed method) or repeated fed batch method(repetitive feed method) for the purpose of production of amino acids,in particular L-lysine. A summary of known culture methods are describedin the textbook by Chmiel (Bioprozesstechnik 1. Einführung in dieBioverfahrenstechnik [Bioprocess Technology 1. Introduction toBioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991) I.B.R.)or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen[Bioreactors and Peripheral Equipment] (Vieweg Verlag,Braunschweig/Wiesbaden, 1994) I.B.R.).

[0106] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981 I.B.R.). Sugars andcarbohydrates, such as e. g. glucose, sucrose, lactose, fructose,maltose, molasses, starch and cellulose, oils and fats, such as e. g.soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids,such as e. g. palmitic acid, stearic acid and linoleic acid, alcohols,such as e. g. glycerol and ethanol, and organic acids, such as e. g.acetic acid, can be used as the source of carbon. These substances canbe used individually or as a mixture. Organic nitrogen-containingcompounds, such as peptones, yeast extract, meat extract, malt extract,corn steep liquor, soy bean flour and urea, or inorganic compounds, suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate, can be used as the source of nitrogen.The sources of nitrogen can be used individually or as a mixture.Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used asthe source of phosphorus. The culture medium must moreover comprisesalts of metals, such as e. g. magnesium sulfate or iron sulfate, whichare necessary for growth. Finally, essential growth substances, such asamino acids and vitamins, can be employed in addition to theabovementioned substances. Suitable precursors can moreover be added tothe culture medium. The starting substances mentioned can be added tothe culture in the form of a single batch, or can be fed in during theculture in a suitable manner.

[0107] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control the pHof the culture. Antifoams, such as e. g. fatty acid polyglycol esters,can be employed to control the development of foam. Suitable substanceshaving a selective action, such as e. g. antibiotics, can be added tothe medium to maintain the stability of plasmids. To maintain aerobicconditions, oxygen or oxygen-containing gas mixtures, such as e. g. air,are introduced into the culture. The temperature of the culture isusually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing iscontinued until a maximum of lysine has formed. This target is usuallyreached within 10 hours to 160 hours.

[0108] The analysis of L-lysine or L-glutamate can be carried out byanion exchange chromatography with subsequent ninhydrin derivatization,as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190)I.B.R.

[0109] The following microorganism has been deposited at the DeutscheSammlung für Mikrorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty:

[0110]Corynebacterium glutamicum strain DSM5715/pJC1cma as DSM 13248

[0111] The method according to the invention is used for thefermentative preparation of amino acids, in particular L-lysine andL-glutamate.

[0112] Legend to the FIGURE

[0113]FIG. 1: Map of the plasmid pJC1cma

[0114] The abbreviations and designations used have the followingmeaning: ORF2,rep: Plasmid-coded replication origin C. glutamicum (ofpHM1519) cma: cma (cyclopropane-mycolic acid synthase) gene from C.glutamicum ATCC13032 Kan: Kanamycin resistance gene BamHI: Cleavage siteof the restriction enzyme BamHI XbaI: Cleavage site of the restrictionenzyme XbaI SalI: Cleavage site of the restriction enzyme SalI PstI:Cleavage site of the restriction enzyme PstI XmaI: Cleavage site of therestriction enzyme XmaI BglII: Cleavage site of the restriction enzymeEglII SphI: Cleavage site of the restriction enzyme SphI EcoRI: Cleavagesite of the restriction enzyme EcoRI

EXAMPLES

[0115] The present invention is explained in more detail in thefollowing with the aid of embodiment examples.

Example 1

[0116] Preparation of a Genomic Cosmid Gene Library From Corynebacteriumglutamicum ATCC 13032

[0117] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 wasisolated as described by Tauch et al. (1995, Plasmid 33:168-179) I.B.R.and partly cleaved with the restriction enzyme Sau3AI (AmershamPharmacia, Freiburg, Germany, Product Description Sau3AI, Code no.27-0913-02) I.B.R. The DNA fragments were dephosphorylated with shrimpalkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany,Product Description SAP, Code no. 1758250) I.B.R. The DNA of the cosmidvector SuperCosl (Wahl et al. (1987) Proceedings of the National Academyof Sciences USA 84:2160-2164 I.B.R.), obtained from Stratagene (LaJolla, USA, Product Description SuperCos1 Cosmid Vektor Kit, Code no.251301), was cleaved with the restriction enzyme XbaI (AmershamPharmacia, Freiburg, Germany, Product Description XbaI, Code no.27-0948-02) and likewise dephosphorylated with shrimp alkalinephosphatase. The cosmid DNA was then cleaved with the restriction enzymeBamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI,Code no. 27-0868-04). The cosmid DNA treated in this manner was mixedwith the treated ATCC 13032 DNA and the batch was treated with T4 DNAligase (Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no. 27-0870-04). The ligation mixture was thenpacked in phages with the aid of Gigapack II XL Packing Extracts(Stratagene, La Jolla, USA, Product Description Gigapack II XL PackingExtract, Code no. 200217). For infection of the E. coli strain NM554(Raleigh et al. 1988, Nucleic Acid Research 16:1563-1575 I.B.R.) thecells were taken up in 10 mM MgSO₄ and mixed with an aliquot of thephage suspension. The infection and titering of the cosmid library werecarried out as described by Sambrook et al. (1989, Molecular Cloning: Alaboratory Manual, Cold Spring Harbor I.B.R.), the cells being platedout on LB agar (Lennox, 1955, Virology, 1:190 I.B.R.) with 100 mg/lampicillin. After incubation overnight at 37° C., recombinant individualclones were selected.

Example 2

[0118] Isolation and Sequencing of the cma Gene

[0119] The cosmid DNA of an individual colony was isolated with theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)in accordance with the manufacturer's instructions and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNAfragments were dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, Product Description SAP,Product No. 1758250). After separation by gel electrophoresis, thecosmid fragments in the size range of 1500 to 2000 bp were isolated withthe QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden,Germany). The DNA of the sequencing vector pZero-1, obtained fromInvitrogen (Groningen, Holland, Product Description Zero BackgroundCloning Kit, Product No. K2500-01) was cleaved with the restrictionenzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product DescriptionBamHI, Product No. 27-0868-04). The ligation of the cosmid fragments inthe sequencing vector pZero-1 was carried out as described by Sambrooket al. (1989, Molecular Cloning: A laboratory Manual, Cold SpringHarbor) I.B.R., the DNA mixture being incubated overnight with T4 ligase(Pharmacia Biotech, Freiburg, Germany). This ligation mixture was thentransformed by means of electroporation (Tauch et al. 1994, FEMSMicrobiol Letters, 123:343-7 I.B.R.) into the E. coli strain DH5αMCR(Grant, 1990, Proceedings of the National Academy of Sciences U.S.A.,87:4645-4649 I.B.R.) and plated out on LB agar (Lennox, 1955, Virology,1:190 I.B.R.) with 50 mg/l zeocin. The plasmid preparation of therecombinant clones was carried out with Biorobot 9600 (Product No.900200, Qiagen, Hilden, Germany). The sequencing was carried out by thedideoxy chain-stopping method of Sanger et al. (1977, Proceedings of theNational Academy of Sciences U.S.A., 74:5463-5467) I.B.R. withmodifications according to Zimmermann et al. (1990, Nucleic AcidsResearch, 18:1067) I.B.R. The “RR dRhodamin Terminator Cycle SequencingKit” from PE Applied Biosystems (Product No. 403044, Weiterstadt,Germany) was used. The separation by gel electrophoresis and analysis ofthe sequencing reaction were carried out in a “Rotiphoresis NFAcrylamide/Bisacrylamide” Gel (29:1) (Product No. A124.1, Roth,Karlsruhe, Germany) with the “ABI Prism 377” sequencer from PE AppliedBiosystems (Weiterstadt, Germany).

[0120] The raw sequence data obtained were then processed using theStaden program package (1986, Nucleic Acids Research, 14:217-231 I.B.R.)version 97-0. The individual sequences of the PZerol derivatives wereassembled to a continuous contig. The computer-assisted coding regionanalysis was prepared with the XNIP program (Staden, 1986, Nucleic AcidsResearch, 14:217-231 I.B.R.). Further analyses were carried out with the“BLAST search program” (Altschul et al., 1997, Nucleic Acids Research,25:3389-3402 I.B.R.), against the non-redundant databank of the“National Center for Biotechnology Information” (NCBI, Bethesda, Md.,USA I.B.R.).

[0121] The resulting nucleotide sequence is shown in SEQ ID No. 1.Analysis of the nucleotide sequence showed an open reading frame of 1353base pairs, which was called the cma gene. The cma gene codes for aprotein of 451 amino acids.

[0122] The following microorganism has been deposited at the DeutscheSammlung füar Mikrorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty:

[0123]Corynebacterium glutamicum strain DSM5715/pJC1cma as DSM 13248

Example 3

[0124] Cloning of the cma gene in the vector pJC1

[0125] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 wasisolated as described by Tauch et al. (1995, Plasmid 33:168-179) I.B.R..A DNA fragment which carries the cma gene was amplified with the aid ofthe polymerase chain reaction. The following primers were used for this:

[0126] 5′-TGC TCT AGA AAA GCA GGT GGG AAA TGG GAC AGT-3′

[0127] 5′-TGC TCT AGA TGG CAG AGC TAG GCG GAC ATA AAT-3′

[0128] The two oligonucleotides carry the sequence for the cleavage siteof the restriction enzyme XbaI (nucleotides underlined). The primersshown were synthesized by MWG Biotech (Ebersberg, Germany) and the PCRreaction was carried out with them by the standard PCR method of Inniset al., (PCR protocol. A guide to methods and applications, 1990,Academic Press) I.B.R. The primers allow amplification of a DNA fragmentof 1653 bp in size, which carries the cma gene from Corynebacteriumglutamicum.

[0129] After separation by gel electrophoresis, the PCR fragment wasisolated from the agarose gel with the QiaExII Gel Extraction Kit(Product No. 20021, Qiagen, Hilden, Germany).

[0130] The PCR fragment obtained in this manner was cleaved completelywith the restriction enzyme XbaI. The cma fragment approx. 1659 bp insize was isolated from the agarose gel with the QiaExII Gel ExtractionKit (Product No. 20021, Qiagen, Hilden, Germany).

[0131] The E. coli-C. glutamicum shuttle vector pJC1 (Cremer et al.,1990, Molecular and General Genetics 220: 478-480) I.B.R. was used asthe vector. This plasmid was also cleaved completely with therestriction enzyme XbaI and then dephosphorylated with shrimp alkalinephosphatase (Roche Diagnostics GmbH, Mannheim, Germany, ProductDescription SAP, Product No. 1758250).

[0132] The cma fragment obtained in this manner was mixed with theprepared vector pJC1 and the batch was treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no. 27-0870-04). The ligation batch was transformedin the E. coli strain DH5α (Hanahan, In: DNA cloning. A practicalapproach. Vol. I. IRL-Press, Oxford, Washington, D.C., USA I.B.R.).Selection of plasmid-carrying cells was made by plating out thetransformation batch on LB agar (Lennox, 1955, Virology, 1:190 I.B.R.)with 50 mg/l kanamycin. After incubation overnight at 37° C.,recombinant individual clones were selected. Plasmid DNA was isolatedfrom a transformant with the Qiaprep Spin Miniprep Kit (Product No.27106, Qiagen, Hilden, Germany) in accordance with the manufacturer'sinstructions and cleaved with the restriction enzyme XbaI to check theplasmid by subsequent agarose gel electrophoresis. The resulting plasmidwas called pJC1cma.

Example 4

[0133] Transformation of the strains DSM5715 and ATCC13032 with theplasmid pJC1cma

[0134] The strains DSM5715 and ATCC13032 were transformed with theplasmid pJC1cma using the electroporation method described by Liebl etal., (FEMS Microbiology Letters, 53:299-303 (1989)) I.B.R. Selection ofthe transformants took place on LBHIS agar comprising 18.5 g/lbrain-heart infusion broth, 0.5M sorbitol, 5 g/l Bacto-tryptone, 2.5 g/lBacto-yeast extract, 5 g/l NaCl and 18 g/l Bacto-agar, which had beensupplemented with 25 mg/l kanamycin. Incubation was carried out for 2days at 33° C.

[0135] Plasmid DNA was isolated from in each case one transformant byconventional methods (Peters-Wendisch et al., 1998, Microbiology 144,915-927 I.B.R.) and cleaved with the restriction endonuclease BamHI, tocheck the plasmid by subsequent agarose gel electrophoresis. Theresulting strains were called DSM5715/pJC1cma and ATCC13032/pJC1cma.

[0136] The following microorganism has been deposited at the DeutscheSammlung für Mikrorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty:

[0137]Corynebacterium glutamicum strain DSM 5715/pJC1cma as DSM 13248

Example 5

[0138] Preparation of L-glutamate with the strain TCC13032/pJC1cma

[0139] The C. glutamicum strain ATCC13032/pJC1cma obtained in example 4was cultured in a nutrient medium suitable for the production ofglutamate and the glutamate content in the culture supernatant wasdetermined.

[0140] For this, the strain was first incubated on an agar plate withthe corresponding antibiotic (brain-heart agar with kanamycin (50 mg/l))for 24 hours at 33° C. Starting from this agar plate culture, apreculture was seeded (10 ml medium in a 100 ml conical flask). Thecomplete medium CgIII (2.5 g/l NaCl, 10 g/l Bacto-peptone, 10 g/lBacto-yeast extract, pH 7.4, 20 g/l Glucose (autoclaved separately) wasused as the medium for the preculture. Kanamycin (25 mg/l) was added tothis. The preculture was incubated for 16 hours at 33° C. at 240 rpm ona shaking machine. A main culture was seeded from this preculture suchthat the initial OD (660 nm) of the main culture was 0.1. Medium CgXIIwas used for the main culture.

[0141] After preculturing in medium CgIII (Keilhauer et al. 1993,Journal of Bacteriology 175:5595-5603 I.B.R.), the strainATCC13032/pJC1cma was cultured in the production medium CgXII (Keilhaueret al. 1993, Journal of Bacteriology 175:5595-5603 I.B.R.)I. 4% glucoseand 50 mg/l kanamycin sulfate were added.

[0142] For induction of the glutamate formation, 20 g Tween 60 (P-1629,Sigma-Aldrich, Deisenhofen, Germany) plus 80 ml water were mixed andautoclaved. About 4 hours after the inoculation, 75 μl of this Tweensolution were added to the culture and culturing was continued.

[0143] Culturing is carried out in a 10 ml volume in a 100 ml conicalflask with baffles. Kanamycin (25 mg/l) was added. Culturing was carriedout at 33° C. and 80% atmospheric humidity.

[0144] After 48 hours, the OD was determined at a measurement wavelengthof 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). Theamount of glutamate formed was determined with an amino acid analyzerfrom Eppendorf-BioTronik (Hamburg, Germany) by ion exchangechromatography and post-column derivatization with ninhydrin detection.

[0145] The result of the experiment is shown in table 1. TABLE 1Glutamate HCl Strain OD (660) mM ATCC13032/pJC1cma 14.7 106 ATCC1303213.8  94

Example 6

[0146] Preparation of Lysine

[0147] The C. glutamicum strain DSM5715/pJC1cma obtained in example 4was cultured in a nutrient medium suitable for the production of lysineand the lysine content in the culture supernatant was determined.

[0148] For this, the strain was first incubated on an agar plate withthe corresponding antibiotic (brain-heart agar with kanamycin (50 mg/l))for 24 hours at 33° C. Starting from this agar plate culture, apreculture was seeded (10 ml medium in a 100 ml conical flask). Thecomplete medium CgIII (2.5 g/l NaCl, 10 g/l Bacto-peptone, 10 g/lBacto-yeast extract, pH7.4, 20 g/l Glucose (autoclaved separately) wasused as the medium for the preculture. Kanamycin (25 mg/l) was added tothis. The preculture was incubated for 16 hours at 33° C. at 240 rpm ona shaking machine. A main culture was seeded from this preculture suchthat the initial OD (660 nm) of the main culture was 0.1. Medium MM wasused for the main culture.

[0149] Medium MM CSL (corn steep liquor) 5 g/l MOPS(morpholinopropanesulfonic 20 g/l acid) Glucose (autoclaved separately)50 g/l (NH₄)₂SO₄ 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O 1.0 g/l CaCl₂ * 2H₂O 10 mg/l FeSO₄ * 7 H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin(sterile-filtered) 0.3 mg/l Thiamine * HCl (sterile-filtered) 0.2 mg/lL-Leucine 0.1 g/l CaCO₃ 25 g/l

[0150] The CSL, MOPS and the salt solution were brought to pH 7 withaqueous ammonia and autoclaved. The sterile substrate and vitaminsolutions were then added, as well as the CaCO₃ autoclaved in the drystate.

[0151] Culturing is carried out in a 10 ml volume in a 100 ml conicalflask with baffles. Kanamycin (25 μg/l) was added. Culturing was carriedout at 33° C. and 80% atmospheric humidity.

[0152] After 24 hours, the OD was determined at a measurement wavelengthof 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). Theamount of lysine formed was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivatization with ninhydrin detection.

[0153] The result of the experiment is shown in table 1. TABLE 1 LysineHCl Strain OD (660) g/l DSM5715/pJC1cma 12.47 8.51 DSM5715 11.90 7.80

[0154]

1 4 1 1801 DNA Corynebacterium glutamicum CDS (248)..(1600) 1 ctccgatgcaccagagggag catttaacag agaaatcgaa cgcgtcatgg cagagctagg 60 cggacataaatcgctgtact ccgaagcgtt ctacaccagg gaagactttg aaaaacttta 120 tggcggaaccatcccggcgc tgctaaaaaa gcagtgggat ccccacagcc gattccccgg 180 tttgtatgaaaagacagtaa aaggcgccta ggatcgctca ctgtaggtag aggcttgtgg 240 tcactac ttgtgg cca cat ttt aaa aaa atg cac aag aag aga aag caa 289 Leu Trp Pro HisPhe Lys Lys Met His Lys Lys Arg Lys Gln 1 5 10 agc att atg agt aac gccgta gcg cag gac ctc atg acc atc gcc gac 337 Ser Ile Met Ser Asn Ala ValAla Gln Asp Leu Met Thr Ile Ala Asp 15 20 25 30 atc gtc gag gcc acg accact gca ccc atc cca ttc cac atc act gcc 385 Ile Val Glu Ala Thr Thr ThrAla Pro Ile Pro Phe His Ile Thr Ala 35 40 45 ttc gat gga agc ttc act ggccct gaa gat gct ccc tac cag ctg ttt 433 Phe Asp Gly Ser Phe Thr Gly ProGlu Asp Ala Pro Tyr Gln Leu Phe 50 55 60 gtt gcc aac acg gat gca gta tcctac atc gca aca gcg cca gga gat 481 Val Ala Asn Thr Asp Ala Val Ser TyrIle Ala Thr Ala Pro Gly Asp 65 70 75 ttg ggt ttg gca cgt gcc tac ctc atggga gac ctc atc gtg gaa ggt 529 Leu Gly Leu Ala Arg Ala Tyr Leu Met GlyAsp Leu Ile Val Glu Gly 80 85 90 gag cat ccc ggc cat cct tat ggg atc tttgat gcg ttg aag gag ttc 577 Glu His Pro Gly His Pro Tyr Gly Ile Phe AspAla Leu Lys Glu Phe 95 100 105 110 tac cgc tgc ttc aaa cgc cca gat gcatcc acc acc ttg cag atc atg 625 Tyr Arg Cys Phe Lys Arg Pro Asp Ala SerThr Thr Leu Gln Ile Met 115 120 125 tgg act ctg cgg aaa atg aat gcc ttaaaa ttc cag gaa att cca cca 673 Trp Thr Leu Arg Lys Met Asn Ala Leu LysPhe Gln Glu Ile Pro Pro 130 135 140 atg gaa caa gcc cct gca tgg cgt aaagca ctg atc aac ggg cta gca 721 Met Glu Gln Ala Pro Ala Trp Arg Lys AlaLeu Ile Asn Gly Leu Ala 145 150 155 tcc agg cac tcg aaa tcc cgc gac aagaaa gcc att agc tac cac tac 769 Ser Arg His Ser Lys Ser Arg Asp Lys LysAla Ile Ser Tyr His Tyr 160 165 170 gac gtg ggc aat gag ttc tac tcc ctgttt tta gat gat tcc atg acc 817 Asp Val Gly Asn Glu Phe Tyr Ser Leu PheLeu Asp Asp Ser Met Thr 175 180 185 190 tat acc tgc gcg tat tat cca acgcca gaa tca agt ttg gaa gaa gcc 865 Tyr Thr Cys Ala Tyr Tyr Pro Thr ProGlu Ser Ser Leu Glu Glu Ala 195 200 205 caa gaa aac aaa tac cgc ctc atcttt gaa aaa ctg cgt ctg aaa gaa 913 Gln Glu Asn Lys Tyr Arg Leu Ile PheGlu Lys Leu Arg Leu Lys Glu 210 215 220 ggc gat cgc ctc cta gac gtg ggatgc ggt tgg gga ggc atg gtc cgc 961 Gly Asp Arg Leu Leu Asp Val Gly CysGly Trp Gly Gly Met Val Arg 225 230 235 tac gcc gcc aaa cac ggt gtg aaagcc atc gga gtt acg ctg tct gaa 1009 Tyr Ala Ala Lys His Gly Val Lys AlaIle Gly Val Thr Leu Ser Glu 240 245 250 cag caa tat gag tgg ggt caa gcagag atc aaa cgc caa ggt ttg gaa 1057 Gln Gln Tyr Glu Trp Gly Gln Ala GluIle Lys Arg Gln Gly Leu Glu 255 260 265 270 gac ctc gcg gaa att cgc ttcatg gat tac cgc gat gtt cca gaa act 1105 Asp Leu Ala Glu Ile Arg Phe MetAsp Tyr Arg Asp Val Pro Glu Thr 275 280 285 gga ttc gat gcg atc tca gcaatc ggc atc att gaa cac atc ggt gtg 1153 Gly Phe Asp Ala Ile Ser Ala IleGly Ile Ile Glu His Ile Gly Val 290 295 300 aac aac tat ccc gac tac tttgaa ttg ctc agc agc aaa ctc aaa aca 1201 Asn Asn Tyr Pro Asp Tyr Phe GluLeu Leu Ser Ser Lys Leu Lys Thr 305 310 315 ggc gga ctg atg ctc aac cacagc atc acc tac cca gac aac cgc ccc 1249 Gly Gly Leu Met Leu Asn His SerIle Thr Tyr Pro Asp Asn Arg Pro 320 325 330 cgc cac gca ggt gca ttt attgat cgc tac att ttc ccc gac ggt gaa 1297 Arg His Ala Gly Ala Phe Ile AspArg Tyr Ile Phe Pro Asp Gly Glu 335 340 345 350 ctc act ggc tct ggc accctg atc aag cac atg cag gac aac ggt ttc 1345 Leu Thr Gly Ser Gly Thr LeuIle Lys His Met Gln Asp Asn Gly Phe 355 360 365 gaa gtg ctg cac gaa gaaaac ctc cgc ttt gat tac caa cgc acc ctg 1393 Glu Val Leu His Glu Glu AsnLeu Arg Phe Asp Tyr Gln Arg Thr Leu 370 375 380 cac gcg tgg tgc gaa aacctc aaa gaa aat tgg gag gaa gca gtt gaa 1441 His Ala Trp Cys Glu Asn LeuLys Glu Asn Trp Glu Glu Ala Val Glu 385 390 395 ctc gcc ggt gaa ccc actgca cga ctc ttt ggc ctg tac atg gca ggt 1489 Leu Ala Gly Glu Pro Thr AlaArg Leu Phe Gly Leu Tyr Met Ala Gly 400 405 410 tcg gaa tgg gga ttt gcccac aac atc gtc cag ctg cac caa gta ctg 1537 Ser Glu Trp Gly Phe Ala HisAsn Ile Val Gln Leu His Gln Val Leu 415 420 425 430 ggt gtg aaa ctc gatgag cag gga agt cgc gga gaa gtt cct gaa aga 1585 Gly Val Lys Leu Asp GluGln Gly Ser Arg Gly Glu Val Pro Glu Arg 435 440 445 atg tgg tgg act atctaaagaaaca atgtttcttt ttaaggttcc ctaggggcag 1640 Met Trp Trp Thr Ile 450gagttaattg taggtaaact cgactagcaa ttagcgactg tcccatttcc cacctgcttt 1700tgcggatttc aacactttca ggatgcatct gtaactatgc ccatgccaca ccccatgttt 1760atcgatgtct ccttcccacc cgcggagacc tgtagttaac c 1801 2 451 PRTCorynebacterium glutamicum 2 Leu Trp Pro His Phe Lys Lys Met His Lys LysArg Lys Gln Ser Ile 1 5 10 15 Met Ser Asn Ala Val Ala Gln Asp Leu MetThr Ile Ala Asp Ile Val 20 25 30 Glu Ala Thr Thr Thr Ala Pro Ile Pro PheHis Ile Thr Ala Phe Asp 35 40 45 Gly Ser Phe Thr Gly Pro Glu Asp Ala ProTyr Gln Leu Phe Val Ala 50 55 60 Asn Thr Asp Ala Val Ser Tyr Ile Ala ThrAla Pro Gly Asp Leu Gly 65 70 75 80 Leu Ala Arg Ala Tyr Leu Met Gly AspLeu Ile Val Glu Gly Glu His 85 90 95 Pro Gly His Pro Tyr Gly Ile Phe AspAla Leu Lys Glu Phe Tyr Arg 100 105 110 Cys Phe Lys Arg Pro Asp Ala SerThr Thr Leu Gln Ile Met Trp Thr 115 120 125 Leu Arg Lys Met Asn Ala LeuLys Phe Gln Glu Ile Pro Pro Met Glu 130 135 140 Gln Ala Pro Ala Trp ArgLys Ala Leu Ile Asn Gly Leu Ala Ser Arg 145 150 155 160 His Ser Lys SerArg Asp Lys Lys Ala Ile Ser Tyr His Tyr Asp Val 165 170 175 Gly Asn GluPhe Tyr Ser Leu Phe Leu Asp Asp Ser Met Thr Tyr Thr 180 185 190 Cys AlaTyr Tyr Pro Thr Pro Glu Ser Ser Leu Glu Glu Ala Gln Glu 195 200 205 AsnLys Tyr Arg Leu Ile Phe Glu Lys Leu Arg Leu Lys Glu Gly Asp 210 215 220Arg Leu Leu Asp Val Gly Cys Gly Trp Gly Gly Met Val Arg Tyr Ala 225 230235 240 Ala Lys His Gly Val Lys Ala Ile Gly Val Thr Leu Ser Glu Gln Gln245 250 255 Tyr Glu Trp Gly Gln Ala Glu Ile Lys Arg Gln Gly Leu Glu AspLeu 260 265 270 Ala Glu Ile Arg Phe Met Asp Tyr Arg Asp Val Pro Glu ThrGly Phe 275 280 285 Asp Ala Ile Ser Ala Ile Gly Ile Ile Glu His Ile GlyVal Asn Asn 290 295 300 Tyr Pro Asp Tyr Phe Glu Leu Leu Ser Ser Lys LeuLys Thr Gly Gly 305 310 315 320 Leu Met Leu Asn His Ser Ile Thr Tyr ProAsp Asn Arg Pro Arg His 325 330 335 Ala Gly Ala Phe Ile Asp Arg Tyr IlePhe Pro Asp Gly Glu Leu Thr 340 345 350 Gly Ser Gly Thr Leu Ile Lys HisMet Gln Asp Asn Gly Phe Glu Val 355 360 365 Leu His Glu Glu Asn Leu ArgPhe Asp Tyr Gln Arg Thr Leu His Ala 370 375 380 Trp Cys Glu Asn Leu LysGlu Asn Trp Glu Glu Ala Val Glu Leu Ala 385 390 395 400 Gly Glu Pro ThrAla Arg Leu Phe Gly Leu Tyr Met Ala Gly Ser Glu 405 410 415 Trp Gly PheAla His Asn Ile Val Gln Leu His Gln Val Leu Gly Val 420 425 430 Lys LeuAsp Glu Gln Gly Ser Arg Gly Glu Val Pro Glu Arg Met Trp 435 440 445 TrpThr Ile 450 3 33 DNA Corynebacterium glutamicum 3 tgctctagaa aagcaggtgggaaatgggac agt 33 4 33 DNA Corynebacterium glutamicum 4 tgctctagatggcagagcta ggcggacata aat 33

What is claimed is:
 1. A genetically modified coryneform bacterium,wherein the cma gene, which codes for cyclopropane-mycolic acidsynthase, is amplified.
 2. The genetically modified coryneform bacteriumas claimed in claim 1, wherein the starting bacterium (wild-type) isselected from the group consisting of Corynebacterium glutamicum(ATCC13032), Corynebacterium acetoglutamicum (ATCC15806),Corynebacterium acetoacidophilum (ATCC13870), Corynebacteriumthermoaminogenes (FERM BP-1539), Corynebacterium melassecola(ATCC17965), Brevibacterium flavum (ATCC14067), Brevibacteriumlactofermentum (ATCC13869) and Brevibacterium divaricatum (ATCC14020),or is selected from the group consisting of Corynebacterium glutamicumFERM-P 1709, Brevibacterium flavum FERM-P 1708, Brevibacteriumlactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463,Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicumDSM5715.
 3. The genetically modified coryneform bacterium as claimed inclaim 1, wherein the amplification of the cma gene is carried out byover-expression of the gene.
 4. The genetically modified coryneformbacterium as claimed in claim 3, wherein the cma gene is over-expressedby increasing the number of copies of the gene, by choosing a potentpromoter or a regulation region above the reading frame, by mutation ofthe promoter, the regulation region or the ribosome binding site, byincorporation of a suitable expression cassette above the structuralgene, by incorporation of inducible promoters, by prolonging the life ofthe corresponding mRNA, by a reduced degradation of the proteinsexpressed, or by combination of several of these possibilities.
 5. Agenetically modified coryneform bacterium as claimed claim 1, whereinthe strain is transformed with a plasmid vector and the plasmid vectorcarries the nucleotide sequence which codes for the cma gene.
 6. Agenetically modified coryneform bacterium as claimed in claim 1, whichcorresponds genotypically to the strain Corynebacterium glutamicum DSM13248.
 7. An isolated polynucleotide from coryneform bacteria,comprising a polynucleotide sequence selected from the group consistingof a) a polynucleotide which is homologous to the extent of at least 70%to a polynucleotide which codes for a polypeptide which comprises theamino acid sequence of SEQ ID No. 2, b) a polynucleotide which codes fora polypeptide which comprises an amino acid sequence which is homologousto the extent of at least 70% to the amino acid sequence of SEQ ID No.2, c) a polynucleotide which is complementary to the polynucleotides ofa) or b), and d) a polynucleotide comprising at least 15 successivenucleotides of the polynucleotide sequence of a), b) or c).
 8. Thepolynucleotide as claimed in claim 7, wherein the polynucleotide is arecombinant DNA which is capable of replication in coryneform bacteria.9. The polynucleotide as claimed in claim 7, wherein the polynucleotideis an RNA.
 10. The polynucleotide as claimed in claim 7, wherein the DNAwhich is capable of replication, comprises (i) the nucleotide sequenceshown in SEQ ID no. 1, or (ii) at least one sequence which correspondsto sequence (i) in the context of the degeneration of the genetic code,or (iii) at least one sequence which hybridizes with the sequencecomplementary to sequence (i) or (ii), and optionally (iv) mutations ofneutral function in (i) which lead to homologous amino acids.
 11. Thepolynucleotide sequence as claimed in claim 10, which codes for apolypeptide which has the amino acid sequence SEQ ID No.
 2. 12. A methodfor the fermentative preparation of L-amino acids comprising carryingout the following steps: a) fermenting coryneform bacteria to produceL-amino acids in which at least the cma gene or nucleotide sequenceswhich code for it is amplified.
 13. The method according to claim 12;wherein the cma gene or nucleotide sequences which code for it isamplified by being over-expressed.
 14. The method according to claim 12,further comprising: b) concentrating the L-amino acid in the medium orin the cells of the bacteria.
 15. The method according to claim 14,further comprising: c) isolating the L-amino acid.
 16. The method asclaimed in claim 12, wherein a genetically modified coryneformbacterium, wherein the cma gene, which codes for cyclopropane-mycolicacid synthase, is amplified, is employed.
 17. The method as claimed inclaim 12, wherein further genes which code a protein of the biosynthesispathway of the desired L-amino acid are additionally amplified in thebacteria.
 18. The method as claimed in claim 12, wherein metabolicpathways which reduce the formation of the desired amino acid are atleast partly eliminated in the bacteria.
 19. The method as claimed inclaim 12, wherein the amino acid prepared is L-lysine or L-glutamate.20. The method as claimed in claim 12, wherein for the preparation oflysine or glutamate, bacteria in which at the same time one or moregenes selected from the group consisting of a) the dapA gene which codesfor dihydrodipicolinate synthase, b) the dapE gene which codes forsuccinyl diaminopimelate desuccinylase, c) the lysC gene which codes fora feed-back resistant aspartate kinase, d) the tpi gene which codes fortriose phosphate isomerase, e) the gap gene which codes forglyceraldehyde 3-phosphate dehydrogenase, f) the pgk gene which codesfor 3-phosphoglycerate kinase, g) the pyc gene which codes for pyruvatecarboxylase, h) the mqo gene which codes for malate:quinoneoxidoreductase, and i) the lysE gene which codes for lysine export, isor are amplified at the same time they are fermented.
 21. The method asclaimed in claim 20, wherein said one or more genes is or areoverexpressed at the same time they are fermented.
 22. A method asclaimed in claim 12, wherein for the preparation of L-lysine, bacteriain which one or more genes chosen from the group consisting of a) thepck gene which codes for phosphoenol pyruvate carboxykinase; and b) thepgi gene which codes for glucose 6-phosphate isomerase is attenuated atthe same time are fermented.
 23. A primer which comprises apolynucleotide sequence as claimed in claim 7, or parts thereof, and canproduce DNA of genes which code for cyclopropane-mycolic acid synthaseby the polymerase chain reaction.
 24. A hybridization probe whichcomprises a polynucleotide sequence as claimed in claim 7 and canisolate cDNA or genes which have a high homology with the sequence ofthe cma gene.